There is increasing awareness that many human maladies relate to a malalignment of the immune system with the needs of the host. Failure to eliminate a pathogenic microorganism often stems from immune hyporesponsiveness or inadequate effector action. On the other hand, tissue destruction in the absence of an invading organism often stems from immune over-responsiveness to an autoantigen. Small-molecule drugs have been developed which are powerful non-specific immune enhancers or depressants. But they are blunt instruments for what is really required—a focused modulation of immune reactivity against a few selected target molecules.
One of the challenges in addressing this question is understanding the difference in the underlying mechanisms for immunogenicity and immunotolerance: respectively, the upward and downward modulation of immunological reactivity. In certain contexts, both types of modulation involve an inducing antigen in a complex interaction with antigen presenting cells and T cells.
The mucosal immune system is more biased towards non-responsiveness and tolerization of a foreign antigen than the systemic immune system. Clearly, reaction against all the foreign substances in the diet would deplete the resources of the system. The mechanisms that dampen responsiveness include clearance of food antigens from the portal circulation by Kuppfer cells, and sampling of antigen through M cells of Peyer's patches and mature enterocytes for presentation to the immune system in a tolerogenic context. Lymphocytes and other cells participating in the mucosal immune system secrete a different spectrum of cytokines and bear a different spectrum of surface markers from their counterparts in the systemic immune system.
The mucosal immune system is common to different mucosal sites, including the bronchus, breast and bowel (Bienenstock et al., Adv. Exp. Med. Biol. 107:53, 1978). In contrast, the mucosal and systemic immune systems are partitioned from each other. The tolerogenic response in the gut involves regulatory TGF-β, IL-4, and IFN-γ secreting T cells (Leonilda et al., Cell. Immunol. 157:439, 1994; Zeng-Yu et al., Cellular Immunol. 157:353, 1994). Cells in the efferent vessel of mesenteric lymph nodes preferentially home back to the mucosa, whereas cells in the efferent vessel of peripheral nodes preferentially home back to the periphery. Nevertheless, there is enough cross-over between the systems that a response invoked in the gut is shared into the systemic compartment. Thus, the Sabin polio vaccine induces protection against polio both at the mucosal surface and in the circulation. Tolerance induced against certain antigens presented in the gut can lead to a systemic non-responsiveness against those antigens.
Efforts have been made to take advantage of the mucosal bias towards tolerance as a mode of therapy for specific immunological down-regulation. For reviews of the area, see Thompson et al. (Immunol. Today 11:197, 1990); Weiner (Proc. Natl. Acad Sci USA 91:10762, 1994); MacDonald (Curr. Biol. 4:178, 1994); and Weiner et al. (Annu. Rev. Immunol. 12:809, 1994). Generally, the approach has been to intubate an antigen into the small intestine of animals with an aberrant immune response, in an effort to lower the responsiveness and thereby improve the condition.
International patent publication WO 91/12816 and EP Patent Application EP 666 080 A1 recites treatment of autoimmune diseases by oral administration of autoantigens. The autoantigen is specific for an autoimmune disease and is orally or enterally administered for eliciting suppressor T cells that recognize the autoantigen. International patent publication WO 91/08760 recites treatment of autoimmune diseases by aerosol administration of autoantigen.
International patent publication WO 96/39176 recites the use of oral administration of antigen to suppress both TH1 and TH2 immune responses and suppress antibody production. In working examples, the antigen was fed ad libitum in the drinking water, and dependent claims recite at least 6 doses per day or sustained release.
International patent publication WO 93/16724 recites treatment of autoimmune disease by administering a bystander antigen rather than an autoantigen associated directly with the disease. The bystander antigen elicits release of TGF-β at a locus within the body of mammals, wherein T cells contributing to autoimmune response are found to suppress the T-cells contributing to the disease. International patent publication WO 94/27634 recites cryptic peptides for use in inducing immunologic tolerance. Cryptic epitopes are those determinants in a protein antigen which, due to processing and presentation of the native protein antigen, are not normally revealed to the immune system.
A number of patent disclosures and academic articles recite treatment of particular conditions by tolerizing through a mucosal route. U.S. Pat. No. 5,399,347 and Thompson et al. (Autoimmunity 16:189, 1993) recite methods of treating rheumatoid arthritis with whole Type II collagen administered orally. International patent publication WO 94/27634 and Vrabec et al. (Autoimmunity 12:175, 1992) recite treating autoimmune uveoretinitis by orally administering S antigen. Chen et al. (Science 265:1237, 1994) and Whitachre et al. (J. Immunol. 147:2155, 1991) recite suppression of autoimmune encephalomyelitis by oral administration of myelin basic protein (MBP). Wang et al. (Cell. Immunol. 152:394, 1993) recite oral administration of acetylcholine receptor to orally tolerize against experimental autoimmune myasthenia gravis.
International patent publication WO 94/27634 recites treating type I diabetes by oral administration of insulin. For other experiments in non-obese diabetic (NOD) mice, see Ramiya et al. (Diabetes 44:164A, 1995) and Zhang et al. (Proc. Natl. Acad. Sci. USA 88:10252, 1991). In a virus-induced antigen-specific diabetic model, oral treatment with insulin started 1 week before or 10 days after the initiating viral challenge prevented appearance of hyperglycemia in >50% of the mice. Oral administration was believed not to affect the generation of anti-beta cell cytotoxic T lymphocytes nor infiltration into the pancreas, but less beta cells were destroyed. The majority of lymphocytes in the islets of successfully treated insulin-treated mice produced IL-4, IL-10, and TGF-β, whereas lymphocytes from symptomatic mice produced mainly γ-IFN.
International patent publication WO 92/07581 recites methods and compositions for suppressing allograft rejection in mammals. The graft recipient is administered by oral or aerosol administration of an agent selected from the group consisting of spleen cells, cultured cells, or extracts derived from the donor or MHC antigens.
The aforementioned disclosures typically indicate that the tolerizing antigen must be administered frequently, up to several times a day, or at a dose of over 1 mg/mouse. In order to maintain tolerance, it is generally necessary to administer the antigen on an ongoing basis.
Cholera toxin is a prototype bacterial enterotoxin released by Vibrio cholerae, and induces active electrolite and water secretion from the intestinal epithelium. It is a protein built from a single A subunit of 28,000 mol. wt. and five B subunits of 11,600 mol wit. The B subunits are aggregated in a ring by tight noncovalent bonds; the A subunit is attached to and probably partly inserted in the B pentamer ring through weaker noncovalent interactions. The B subunits are responsible for cell binding, and the A subunit has toxic activity involving modifying the G proteins of the cyclic AMP pathway. The binding activity of the B subunit is towards the ganglioside GM1, which is present on the mucosal surface.
There is an extensive literature regarding the ability of cholera toxin to work as an adjuvant in mucosal vaccines, increasing the level of the immune response against the antigen it is mixed with. See, for example, Elson, Curr. Top. Microbiol. Immunol. 146, 1989; Nedrud et al., J. Immunol. 139:3484, 3492, 1987; and McKenzie et al., J. Immunol. 133:1818, 1984.
More recently, the CTB subunit coupled to certain antigens have been indicated as having a tolerance-inducing effect when administered to a mucosal surface.
International patent publication WO 95/10301 recites an immunological tolerance-inducing agent comprising a mucosa binding molecule coupled to a specific tolerogen. Exemplary mucosa-binding structures were CTB and subunit of heat-labile enterotoxin of E. coli. 
Sun et al. (Proc. Natl. Acad. Sci. USA 93, 7196, 1996) recites CTB as an efficient transmucosal carrier-delivery system. Red blood cells were modified by covalently coupling with GM1, which was then attached with the CTB. HGG was modified by covalently coupling directly to CTB. A single oral administration of a soluble or particulate antigen coupled to CTB enhanced tolerance. Sun et al. (Proc. Natl. Acad. Sci. USA 93, 7196, 1996) recites treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein covalently conjugated to CTB.
International patent publication WO 96/21458 recites collagen-based methods and formulations for treatment of immune system-mediated diseases like autoimmune liver disease, Crohns disease, Goodpasture's syndrome, psoriasis, localized sclerosis, the various manifestations of arthritis, and various localized degradative inflammatory or fibrotic conditions. The compound is administrered linked to a mucosa binding molecule such as CTB.
In the experiments of these publications, the effective ingredient was assembled by conjugating with antigen with either the CTB directly, or with a linker group.